The basis or mechanism for the formation of facial wrinkles is the tensing of the muscles of the epidermis that drag the skin inwards. This muscular tension is the result of hyperactivity of the nerves innervating the facial muscles. Nerve hyperactivity is characterized by the uncontrolled and excessive release of neurotransmitters that excite muscle fibers. Because of this, the molecules that control neuronal exocytosis contribute to relaxing muscular tension, and consequently, to eliminating wrinkles.
Botulinum toxins are a family of bacterial neurotoxins produced by Clostridium Botulinum (1) [see section regarding BIBLIOGRAPHY]. 7 different serotypes are known (serotypes A, B, C, D, E, F and G) with an average molecular weight of 150 kDa. These toxins inhibit acetylcholine exocytosis in the neuromuscular junction (nerve-muscle synapse) of the skeletal muscle (1).
At a molecular level, botulinum toxins are proteases that degrade neuronal proteins involved in the exocytosis mechanism activated by the calcium ion (1-3). For example, botulinum toxin A, the one most commonly used clinically and cosmetically [because of its applications in eliminating facial wrinkles and asymmetry, and to mitigate the symptomatology of spastic diseases], cleaves the neuronal protein SNAP-25. This protein (SNAP-25) plays a key role in neurosecretion, as it is involved in the formation of a protein complex (known as SNARE complex or fusion complex), which directs and controls the release of acetylcholine accumulated in vesicles. The nucleus of said fusion complex is made up of proteins SNAP-25 and syntaxin, located in the presynaptic plasma membrane, and protein synaptobrevin (or VAMP), located in the vesicular plasma membrane (4, 5). The main function of the fusion complex is to bring the vesicle loaded with neurotransmitter (acetylcholine) nearer to the presynaptic plasma membrane and put it in contact with same (4, 5). In this way, in response to an elevated concentration of calcium, the fusion of both plasma membranes is encouraged, thus producing the release of the neurotransmitter. Therefore, said vesicle docking and fusion protein complex (SNARE) is a key target in controlling neurosecretion. Cleaving any of the proteins that make up the fusion complex prevents its assembly, and therefore inhibits vesicle release and neuronal exocytosis.
The power of botulinum toxins and, in particular, serotype A (BOTOX®) to inhibit neurosecretion, as well as their neuronal selectivity (they only act on neurons) is being widely used therapeutically to correct spastic ailments such as dystonias, strabismus, tics, blepharospasm, facial scoliosis, etc. (6-13). botulinum toxin A (botulinum A) is, moreover, an effective agent for eliminating facial wrinkles and asymmetry. In fact, the administration of BOTOX® is the first effective non-surgical therapy to eliminate the signs of aging (6, 7).
Therapeutic and cosmetic treatment with BOTOX® consists of a localized injection of diluted pharmaceutical preparations (botulinum A-hemagglutinin complex, 500 kDa) in the areas where muscular tension is localized. The paralytic effects of the toxin are reversible with an average duration of 6 months (6, 7). The treatment, therefore, requires repeated injections of BOTOX®. The main problem with this treatment is the chance that it may trigger an immune reaction against the pharmaceutical preparation due to the fact that, because of its molecular size, it may be recognized by the patient's immune system. The appearance of antibodies against botulinum A is a serious problem, as it contributes to a clear decrease in the treatment's effectiveness (6-13). This loss of effectiveness in treatment with BOTOX® means the need to increase the preparation's concentration level in later treatments, which in turn produces a potentiation of the immune response. As an alternative, the use of different botulinum toxin (BoTox) serotypes has been discussed, such as BoTox B, BoTox F and BoTox E. Nevertheless, the application of pharmaceutical preparations with different serotypes cannot be considered a solution to the problem, as sooner or later the immune reaction may once again occur. In addition, treatment with botulinum toxins is expensive, mainly because of the lability and instability of the pharmaceutical preparations containing them.
There is, therefore, a pressing need to develop molecules that imitate the paralytic effects of the botulinum toxins, but with much simpler and more stable molecular structures, which do not cause immune reactions, and whose manufacturing cost is economical. Peptide-type molecules comply with these properties.
Amino acid sequences that inhibit neuronal exocytosis have been described. Specifically, it has been proven that peptides with more than 20 amino acids, deriving from the C-terminal sequence of SNAP-25, block the release of catecholamines from permeabilized chromaffin cells (14). Likewise, peptides deriving from the amino acid sequences of proteins syntaxin and VAMP have been described that can also affect the exocytotic process (15). Although these peptides show biological activity, their later development as cosmetic and/or therapeutic agents has not occurred, most likely due to their size, as this complicates their development as useful therapeutic agents and makes it more expensive. Therefore, there is a need to find molecules of a smaller size that can be applied in cosmetics and medicine.
This invention provides a solution to the existing needs which includes the discovery of smaller amino acid sequences, between 3 and 30 amino acids, deriving from the amino end (N-terminal domain) of protein SNAP-25, which inhibit neuronal exocytosis. In addition, it has been discovered that the simultaneous use of peptides deriving from the amino end and from the carboxyl end (C-terminal domain) of SNAP-25 produces a considerable increase in their inhibitory activity, i.e., there is a potentiation of their activity compared to that shown by individual peptides.
Therefore, one object of this invention is a peptide that has a sequence made up of 3 to 30 adjacent amino acids contained on the amino end of protein SNAP-25, which inhibits neuronal exocytosis.
An additional object of this invention is a nucleic acid that essentially codes for one of the peptides provided by this invention. The plasmids and vectors that contain said nucleic acid (also identified as constructions), as well as the cells transformed with said plasmids or vectors that express a peptide of the invention, also constitute additional objects of this invention.
Another additional object of this invention is a mixture of at least one of the peptides provided by this invention and at least one peptide that has a sequence made up of 3 to 30 adjacent amino acids contained on the carboxyl end of protein SNAP-25.
Another additional object of this invention is a cosmetic composition that includes at least one of the peptides provided by this invention. The use of the peptides provided by this invention in the preparation of said cosmetic composition, as well as the method of cosmetic treatment that includes the application of said cosmetic composition, constitute additional objects of this invention.
Another additional object of this invention is a pharmaceutical composition that includes at least one of the peptides provided by this invention, or alternatively, a vector containing a nucleic acid that codes for one of the peptides of the invention. The use of the peptides and vectors (constructions) provided by this invention in the preparation of said pharmaceutical compositions, as well as the method of treating humans or animals encompassed by the application of said cosmetic composition, constitute additional objects of this invention.
Another additional object of this invention is a combination of drugs that includes at least one of the peptides provided by this invention, along with, at least, one drug intended for a second therapeutic target which may be the same as or different from the therapeutic target at which the peptide provided by this invention is aimed.